Although oxygenated fatty acids and oxidized phospholipids are critical signaling molecules (and/or biomarkers) in acute lung injury (ALI), their extraordinary diversity along with limitations in biochemical methodologies have prohibited their systematic study. We recently described new methodology of oxidative lipidomics that combines the use of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI- MS). We now propose to develop a new enabling technology of imaging mass spectrometry (IMS) for oxidative lipidomics that will resolve issues of spatial confinements of peroxidation reactions in lipids in regions of the lung and ultimately near limits of individual pulmonary cells. Accordingly, the overall aim of this proposal is to: develop IMS of oxidative lipidomics and define spatial distribution of oxidized, oxidizable and non-oxidizable phospholipids - particularly of anionic cardiolipin (CL) and phosphatidylserine (PS) - in lungs of intact mice after oxidant (H2O2-generating glucose oxidase)-mediated ALI. To this end, we will utilize multiple approaches of increasing power in resolution: a) Matrix assisted laser desorption ionization (MALDI)-IMS-TOF-TOF(~50mm); b) the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance MS (~20mm resolution, Bruker Daltonics SolariX, Billerica, MA); and c) high spatial resolving power of nano-scale matrices combined with MALDI-post-ionization mobility-orthogonal Time of Flight MS (~10mm Ionwerks, Inc, NIH NIDA) and oversampling-stepping (<10mm). Serial sections will be used for: a) quantitative measures of oxidative lipidomics via LC-ESI-MS; and b) fluorescence immunohistochemistry that will be co-registered with IMS spectra to reveal structure and function of lung in ALI. The use of mitochondrial targeted antioxidants will serve as a validation of this technology and expand application of such imaging to metabolomics and molecular pharmacology. This proposal will establish a new enabling technology (IMS) for oxidative lipidomics that will: a) resolve issues of spatial confinements of peroxidation in lung that cannot be readily examined otherwise; b) provide a panoramic snap-shot of hundreds of signals simultaneously; c) be informative about oxidation of critical phospholipids (CL and PS) that are early signaling molecules in cell death (apoptosis) and inflammation (clearance of apoptotic cells); and d) be used in a metabolomic mode to reveal molecular pharmacological information on disposition of small molecule protectants in ALI.